University of New Hampshire University of New Hampshire Scholars' Repository
Doctoral Dissertations Student Scholarship
Spring 1973
PURIFICATION AND CHARACTERIZATION OF HEXOSE-OXIDASE FROM THE RED ALGA, CHONDRUS CRISPUS
JAMES DENIS SULLIVAN JR.
Follow this and additional works at: https://scholars.unh.edu/dissertation
Recommended Citation SULLIVAN, JAMES DENIS JR., "PURIFICATION AND CHARACTERIZATION OF HEXOSE-OXIDASE FROM THE RED ALGA, CHONDRUS CRISPUS" (1973). Doctoral Dissertations. 1023. https://scholars.unh.edu/dissertation/1023
This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. INFORMATION TO USERS
This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted.
The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction.
1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity.
2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image o f the page in the adjacent frame.
3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete.
4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced.
5. PLEASE NOTE: Some pages may have indistinct print. Filmed as received.
Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I 73-25,784 I f SULLIVAN, Jr., James Denis, 1942- I PURIFICATION AND CHARACTERIZATION OF HEXDSE ■ OXIDASE FROM THE RED ALGA CHONDRUS CRISPUS.
| University of New Hampshire, Ph.D., 1973 Biochemistry
j University Microfilms, A XEROX Company, Ann Arbor, Michigan
© 1973
JAMES DENIS SULLIVAN, JR .
ALL RIGHTS RESERVED
i THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. J
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PURIFICATION AND CHARACTERIZATION OF HEXOSE OXIDASE FROM THE RED ALGA CHONDRUS CRISPUS
fcy JAMES D. SULLIVAN, JR.
B.S., University of Rhode Island, 1965
M.S., University of Rhode Island, 1967
A THESIS
Submitted to the University of New Hampshire
In Partial Fulfillment of
The Requirements for the Degree of
Doctor of Philosophy Graduate School
Department of Biochemistry
April 1973
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This thesis has been examined and approved.
A. Thesi^ director, Miyoshi Ikawa, Prof. of Biochemistry
Douglas Gr. Routletf^ Prof. of Biochemistry & Plant Science
Gerald 1. ICLip£enstein, Assoc. Prof. of Biochemistry
Paul R. Jones, Pr^£. of Chemistry
Arthur C. Mathieson, Assoc. Prof. of Botany
/YX3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS
Sincere appreciation is extended to Dr. Ikawa for
his helpful suggestions and guidance during this inves
tigation. I also wish to thank Dr. C.L. Grant for atomic
absorption analyses. Part of this study was supported by
U.S. Public Health Service Grant EC-0029^. As a recip
ient of an NDEA fellowship for two years, I also am
grateful to the U.S. Department of Health, Education,
and Welfare (Office of Education). Thanks are also
given to the members of my graduate committee, in
addition to Dr. Ikawa, for reviewing this thesis.
Finally, to my wife Peggy and parents, sincere thanks
are given for their encouragement.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS
LIST OF TABLES ...... vi
LIST OF FIGURES . .. .vii
ABSTRACT ...... ix
I. Introduction ...... 1
II. Materials and Methods ...... 5
1. Determination of Protein and Carbohydrate . 5
2. Copper determination ...... « 5
3. Disc gel electrophoresis ...... 11
4. Assay of hexose oxidase ...... 11
5. Collection, drying, and grinding
of Chondrus crispus ...... 1^
6. Extraction of Chondrus crispus ...... 1^
7. Purification of the C. crispus enzyme... 15
III. Results ...... 22
1. Initial studies on the growth-inhibitory
substance in Chondrus crispus: Clues to
its nature and mode of action ...... 22
2. Evidence for algal origin of hexose
oxidase in C. crispus ...... 25
3. Purification of Chondrus hexose oxidase ... 26
k. Composition and molecular weight of
Chondrus hexose oxidase ...... 28
5. Properties of Chondrus hexose oxidase .... 3&
6. Products of Chondrus hexose oxidase ...... 4>5
iv
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV. Discussion...... 5°
V. Summary and Conclusions ...... 56
VI. Bibliography ...... 58 VII. Appendix ...... 62
v
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L IS T OF TABLES
1. Purification of hexose oxidase from
Chondrus crispus ...... 27
2. Amino acid composition of Chondrus
hexose oxidase ...... 32
3. Substrate specificity of Chondrus
hexose oxidase ...... 43
4. Comparison of substrate specificity of
Euthora and Chondrus enzymes and
glucose oxidase ...... 44
5. Effect of various inhibitors on
Chondrus hexose oxidase ...... 46
6. Paper chromatography of products from
the Chondrus hexose oxidase reaction ...... 48
7. A comparison of properties for
various "glucose oxidases" ...... 51
1A. Growth-inhibitory activity of some toxins
and inhibitors on Chlorella strains ...... 63
2A. Effect of several pesticides on Chlorella
strains ...... 65
3Ao Effect of various compounds on
Chlorella pyrenoidosa (UNH strain) ...... 66
4a . Effect of steroidal compounds on
Chlorella pyrenoidosa...... 67
vi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L IS T OF FIGURES
1. Standard curve for determination of protein
by Lowry method using bovine serum albumin .... 7
2. Standard curve for determination of car
bohydrate by anthrone method using
D-galactose ...... 9
3. Standard curve for determination of
copper using dithizone method ...... 10
4. Standard curve for determination of
units of enzyme activity...... 13
5. DEAE-cellulose chromatography of
Chondrus hexose oxidase ...... 18
6. Gel filtration of Chondrus hexose
oxidase on Sephadex G-200 ...... 21
7. Effect of the red alga Chondrus crispus
on Chlorella pyrenoidosa (UNH strain) ...... 24
8. Disc gel electrophoresis of purified
Chondrus hexose oxidase ...... 30
9. Visible spectrum of purified Chondrus enzyme .. 35
10. Relationship of elution volume to
molecular weight for several protein
standards and Chondrus hexose oxidase on
a column of Sephadex G-200 (2.5 x 43 cm) .... 37
11. Effect of pH on enzyme activity ...... 39
vii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES (cont'd)
12. Effect of incubation temperature
on enzyme activity ...... 40
13. Heat stability of Chondrus hexose oxidase ...... 41
14. Effect of substrate concentration
on reaction velocity ...... 42
viii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT
PURIFICATION AND CHARACTERIZATION
OF HEXOSE OXIDASE FROM THE RED ALGA CHONDRUS CRISPUS
by
James D. Sullivan, Jr.
Hexose oxidase (D-hexose:02 oxidoreductase,
EC 1.1.3.5) has been isolated, purified, and characterized
from the red alga, Chondrus crispus. The enzyme oxidizes
the following substrates: D-glucose, D-galactose, maltose,
lactose, and cellobiose. Products of the reaction include
hydrogen peroxide and the sugar lactone. The production
of hydrogen peroxide has been shown responsible for the
growth-inhibitory effect of C. crispus to Chlorella
pyrenoidosa. Optimum temperature and pH for the Chondrus
hexose oxidase reaction are 25°C and 6 .3, respectively. A
molecular weight of approximately 130,000 has been deter
mined by gel filtration on Sephadex G-200. The purified
enzyme contains ca. 0.6% copper which represents about 12
gram atoms Cu per mole of enzyme of molecular weight
130,000. Chondrus hexose oxidase is a glycoprotein con
taining ca. 70% carbohydrate which consists mainly of
galactose and xylose. Flavin adenine dinucleotide, the
coenzyme of glucose oxidase, is not detectable in the
Chondrus enzyme. Resistance to proteolytic digestion with
pepsin and trypsin is found. Approximately 11$ of the
ix
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. original activity is recoverable following a purification
procedure involving n-butanol treatment, ammonium sulfate
precipitation, DEAE-cellulose chromatography, pepsin-
trypsin digestion, and gel filtration on Sephadex G-200.
The purified enzyme shows a single band staining with
Coomassie blue on disc gel electrophoresis at pH 8.0.
x
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1
INTRODUCTION
The unicellular green alga Chlorella pyrenoidosa
has been shown to be a particularly useful organism for
assaying various toxins of fungal and algal origin (1).
In most cases, toxin-containing paper disks or toxin-
producing organisms when placed on a Chlorella-seeded
agar plate produce a circular zone of inhibition which
appears colorless against a green background. By this
method, numerous compounds at a given concentration or
organisms themselves can easily be screened for toxicity.
Not all species of Chlorella however are equally sensitive
when certain compounds are screened (2). The growth-
inhibitory activity of an extensive list of compounds
against several Chlorella strains is found in the APPENDIX
of this thesis. C. pyrenoidosa (UNH strain) has been
used almost exclusively in this investigation.
Two red algae, Chondrus crispus and Euthora cristata, have been shown to inhibit the growth of C.
pyrenoidosa (1). The objective of the study reported
herein has been the isolation, purification and character
ization of the causative substance in Chondrus. Some
preliminary studies have been done with E. cristata
although not as detailed due to the limited quantity
available.
The compound which appears to be directly involved
in the growth-inhibitory response is hydrogen peroxide.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2
The production of H202 has been attributed to the action of a "glucose oxidase" in C. crispus on glucose which is a
constituent of the Chlorella growth medium. A procedure
for purifying this enzyme has been developed and informa
tion on its properties gathered.
An enzyme, referred to as carbohydrate oxidase
and quite similar in properties to the Chondrus enzyme,
has been isolated and partially purified from the red alga
Iridophycus flaccidum (3). This type of enzyme has since
been named D-hexose:02 oxidoreductase (EC 1.1.3.5) or
simply hexose oxidase because a somewhat unusual property
of the Iridophycus enzyme is its wide range of substrate
specificity which includes D-glucose, D-galactose, maltose,
lactose, and cellobiose. This characteristic distinguishes
this enzyme from glucose oxidase (EC 1.1.3.40 which is
highly specific for D-glucose. In addition to being found
in some red algae, "glucose oxidases" are also known to
occur in honey (4-), bacteria (5.6)» fungi (7-10), and
citrus fruits (11). Most of these enzymes could be ex
pected to inhibit the growth of C. pyrenoidosa through
their action on D-glucose which results in H202 production.
The bacterium Malleomyces pseudomallei has been
reported to contain an enzyme with nearly equal specifi
city for D-glucose and D-galactose (5). To what extent,
if any, maltose, lactose, and cellobiose are attacked,
has not been shown. If these disaccharides are oxidized,
it is quite possible the bacterial enzyme closely resembles
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 the Iridophycus enzyme in other properties as well. The
oxidation product of D-glucose in the presence of this
enzyme is D-gluconic acid with the lactone occurring as an
intermediate in the reaction. The formation of the aldonic
acid (or lactone) appears to he quite typical of "glucose
oxidases".
Species of Aspergillus and Benicillium are known
to contain glucose oxidase which has heen characterized
as a flavin-containing enzyme with a molecular weight
between 150,000 and 160,000. The main characteristic of
this enzyme is the presence of flavin adenine dinucleotide
(FAD). Another enzyme containing FAD is lactose dehydro
genase which oxidizes in addition to lactose: D-glucose,
D-galactose, D-mannose, L-arabinose, D-ribose, D-xylose,
and maltose. Lactobionolactone is the product of the
enzymatic oxidation of lactose (6). In several other
partially purified "glucose oxidases" FAD has not been
detected (3-5, 9, 11). ' A coenzyme other than FAD has been found in an
enzyme bearing some relation to glucose oxidase. This
particular enzyme is galactose oxidase which contains 1
gram atom copper per mole of enzyme of molecular weight
75,000 (12). Although H2O2 is produced in the reaction,
the oxidation of D-galactose occurs at the C-6 position
giving rise to a hexodialdose (13) rather than C-l
oxidation, which in the case of glucose oxidase results
in the formation of the aldonic acid (or lactone).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4
Purified galactose oxidase does not oxidize D-glucose at
a detectable rate (13)• Glucose oxidase and galactose
oxidase are highly substrate specific, produce H202 , and
contain as coenzyme FAD and copper, respectively. Copper
has not previously been reported as a constituent of
"glucose oxidases".
C. pyrenoidosa appears to be a good assay organism
against which numerous algae could be screened for "glucose
oxidase" activity. Although not all inhibition may be due
to the action of such an enzyme, the possibility of H202
as the growth-inhibitory substance is quite good. Whether
or not H2o 2 is responsible can be determined by assaying
an aqueous algal extract with the o-dianisidine-peroxidase
system described in the METHODS section. In the presence
of H202 and peroxidase, the chromagen, o-dianisidine is
transformed to a colored product. If this conversion does
not result, something other than H202 may be involved in
the growth-inhibitory response.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 MATERIALS AND METHODS
The following were obtained from commercial
sources: Sephadex G-200 (Pharmacia Fine Chemicals),
Whatman DE 52 DEAE-cellulose (Reeve-Angel), pepsin and
trypsin (Nutritional Biochemicals); Aspergillus niger
glucose oxidase (1100 units/ml), o-dianisidine diHCl, and
peroxidase (Sigma Chemical). Standards for gel filtration
included ribonuclease (Nutritional Biochemicals),, and
myoglobin, chymotrypsinogen, ovalbumin, albumin, gamma
globulin, apoferritin (Schwarz-Mann). Other chemicals
used were of reagent grade.
Determination of Protein and Carbohydrate
Protein was determined by the method of Lowry
et al. (1*0 using bovine serum albumin as the standard
and carbohydrate by the anthrone method (15) using
D-galactose as the standard. The standard curves are
depicted in Figs. 1 and 2, respectively.
Copper Determination
Copper was determined by atomic absorption
spectroscopy and the dithizone method (16). Before
either determination was made, the lyophilized sample
(ca. 10 mg) was wet-ashed with a 3*5 nil mixture containing
3 ml nitric acid and 0.5 ml 35$ perchloric acid and then,
neutralized with ammonium hydroxide (16). With the
dithizone method, a standard curve was obtained using
CuSO^'Sf^O as the source of copper (Fig. 3)*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6
Fig. 1. Standard curve for determination
of protein by Lowry method using bovine
serum albumin.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with perm ission of the copyright owner. Further reproduction prohibited w ithout permission. ithout w prohibited reproduction Further owner. copyright the of ission perm with Reproduced
0.4 uiu s09990UBqaosqv V o o o o o 7 Micrograms of Protein Fig. 2. Standard curve for determination
of carbohydrate by anthrone method using
D-galactose.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9 Micrograms of D-Galactose of Micrograms
uiu 029 30UBqaosq.v
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5
3 2 Micrograms of Copper 1 Pig. 3. Standard curve for determination of copper using 0 dithizone method. 0.35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11
Disc Gel Electrophoresis
The purity of Chondrus hexose oxidase was deter
mined by disc gel electrophoresis. Standard gels (7$)
were run at 5°C and 2 mA per tube, using a Tris-barbital
buffer with a running pH of 8.0 as described by Williams
and Reisfeld (17). Gels were stained with Coomassie blue
(0.25$ in methanol:water:acetic acid, 5:5=1) and destained
electrophoretically with a Canalco gel destainer using 7$
acetic acid.
Assay of Hexose Oxidase
£-dianisidine-peroxidase system:
The procedure used for assaying the Chondrus
enzyme was based on methods given for the assay of glucose
oxidase (4-, 18). The assay mixture consisted of the follow
ing: 1.5 rnl glucose (0.1 M in 0.1 M sodium phosphate pH
6.3)» 1.2 ml sodium phosphate buffer pH 6.3, 0.1 ml
o-dianisidine diHCl (3.0 mg/ml in water), 0.1 ml peroxidase
(0.1 mg/ml in sodium phosphate buffer), and 0.1 ml enzyme
solution. The mixture was incubated at 25°C for 15 minutes.
The reaction was stopped by adding 1 drop of conc. HC1,
and the abosrbance read at 402 nm. A standard curve was
constructed using varying concentrations of hydrogen per
oxide (0-3.0 jAg/ml) in place of enzyme solution (Fig. 4).
One enzyme unit was defined as that amount of enzyme which
catalyzes the production of 10 J jjimole H202 per minute at 25°C, pH 6.3» and substrate concentration of 0.05 M.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12
Fig. Standard curve for determination
of units of enzyme activity.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 Micrograms Micrograms H£0. of
O O n 0 0 O- uni HOii V& eotreqaosqv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 4 Chlorella assays Assays with Chlorella pyrenoidosa (UNH strain) were done in buffered agar plates as previously described (1). To a Chlorella-seeded plate was added a sterile paper disk (Difco) which contained approximately 20 pi of test solution. After several days exposure to continuous fluorescent lighting, zones of inhibition appeared as colorless areas against a green background. The zone diameter minus the disk diameter was referred to as the 'net inhibition zone'. Collection. Drying, and Grinding of Chondrus crispus Chondrus crispus was collected year-round in the inter-tidal zone at Rye Beach, New Hampshire. Freshly collected fronds were taken to the laboratory as soon as possible where they were washed with cold tap water, blotted, and allowed to air-dry at room temperature for several days. Air-dried fronds were ground to a powder (#16 mesh) with a Wiley mill and then stored in a freezer prior to extraction. Extraction of Chondrus crispus To a 100 g sample of air-dried ground C, crispus fronds was added 1000 ml of 0.1 M sodium phosphate buffer pH 6.8. The mixture was kept at 5°C for 1-2 days during which time it was shaken periodically by hand. The mixture was then filtered through cheesecloth using gentle suction and the filtrate with washings were collected in an Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 ice-co;oled flask. The residue which still contained some activity was discarded. The extract was further clarified by centrifugation at 20,000 x g for 30 minutes. The bright, red-orange supernate was recovered and purified by the following procedure. Purification of the C. crispus Enzyme All steps during purification were carried out at 0-5°C unless stated otherwise. Step 1^. n-Butanol extraction. The 20,000 x g supernate was mixed with an equal volume of n-butanol and after standing for several minutes, the mixture was centri fuged at 10,000 x g for 30 minutes, This treatment, as described by Leibo and Jones (19), caused a deposition of the unwanted photosynthetic pigment phycocyanin at the interface. The aqueous phase, red-orange in color due to the presence of phycoerythrin, was removed and the butanol fraction discarded. Step 2. Ammonium sulfate precipitation. To the butanol-treated extract was slowly added with shaking, solid ammonium sulfate at ^5 g/100 ml. After standing for several hours, the contents were centrifuged at 12,000 x g for 20 minutes. The precipitate was dissolved with stirring in 50-100 nil of 0.01 M sodium phosphate buffer pH 6.8. This solution was transferred to dialysis tubing and dialyzed against a minimum of four 2-liter changes of distilled water over a period of 2-3 days. Insoluble material in the retentate was removed by centrifugation at Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 10,000 x g for 10 minutes. To the supernate was added sodium phosphate sufficient to make the solution 0.1 M pH 6.8. Step J.. DEAE-cellulose chromatography. A DEAE- cellulose column (1.5 x 12 cm) was prepared using 10 g of Whatman DE 52 ion exchange cellulose and equilibrated with 0.1 M sodium phosphate pH 6.8. The sample which had been equilibrated with the same buffer was applied to the column. Following sample application, the column was washed with 5°° ral of "the same buffer used for eq&ilibration. Stepwise addition of this buffer containing 0.3 M NaCl resulted in desorption of the Chondrus enzyme from the column. Fractions from the DE 52 column (Fig. 5) showing activity in the Chlorella assay were pooled and dialyzed against several 1-liter changes of distilled water overnight. Step 4. Pepsin-trypsin treatment. The retentate was adjusted to pH 3*5 with dilute HC1 (final volume ca. 80 ml). To the acidified solution was added 20 mg pepsin (3X crystallized) and the mixture incubated with shaking for 5 hours at 37°C. The reaction was stopped by adjusting the pH to 6.8 with dilute NaOH. Sodium phosphate was added to the digest to make the solution 0.01 M pH 6.8 with respect to phosphate. The mixture was then treated with 20 mg trypsin (2X crystallized) with shaking for 5 hours at 37°C. Following this treatment, the digest was freeze-dried. Step Gel filtration. The lyophilized digest Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 Fig. 5. DEAE-cellulose chromatography of Chondrus hexose oxidase. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 Elution Elution Volume (ml) o o VP\ o o o (rnui) euoz uoTq.xqTiiui q.9M Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was suspended in 3 ml of distilled water and applied to a column (2.5 x 96 cm) of Sephadex G-200 and the column developed with 0.1 HI sodium phosphate pH 6.8. Fractions showing activity in the Chlorella assay (Fig. 6). were pooled, dialyzed extensively against distilled water and freeze-dried. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 Fig. 6. Gel filtration of Chondrus hexose oxidase on Sephadex G-200. Vo=void volume determined with Blue Dextran (MW 2 x 106 ). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (0 ----0) (UIUI) 0UOZ UOXqxqxqUI Q.0M 21 00 VO CVJ O 00 VO ^ C V J O Elution Volume (ml) o o (» -■ e ) uiu Q8H V& eouBqjtosqv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22 RESULTS Initial Studies on The Growth-Inhibitory Substance in Chondrus crispus: Clues to Its Nature and Mode 6f Action The effect of Chondrus crispus on Chlorella pyrenoidosa is shown in Fig. 7. Identical results are found with Euthora cristata. The effect of inhibition appears as a colorless area against a green background. Other algae which have also been found to inhibit the growth of C. pyrenoidosa (UNH strain) although not as greatly are Polysiphonia nigrescens and Membranoptera alata. Algae which are not inhibitory include Gigartina stellata, Sacchoriza dermatodea. Polysiphonia nigra. P. elongata. P. fibrillosa. P. urceolata. A'nnfeltia plicata. Ceramium strictum. and Lomentaria orcadensis. Whether P. nigrescens and/or M. alata contain a growth- inhibitory substance similar to that found in C, crispus and E. cristata is not known. Some of the properties obtained for the active principle in C. crispus at the beginning of this investi gation were the following: heat labile, sensitive to extreme pH, resistant to pepsin and trypsin, resistant to DNase and RNase, and high molecular weight (non-dialyzable) which were somewhat suggestive of a protein. However, the large zones of inhibition were difficult to explain in terms of diffusion of such a large substance as a protein. For this reason, the possibility of an enzymatic reaction Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 Fig. 7. Effect of the red alga Chondrus crispus on Chlorella pyrenoidosa (UNH strain). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 which resulted in a toxic end-product low enough in molecular weight to account for large diffusion zones was considered. An experiment designed to test this hy pothesis involved placing both ground C. crispus fronds and also a dialyzed aqueous extract, each contained in dialysis tubing, on Chlorella-seeded agar plates. Large zones of inhibition were found in both instances which indicated the diffusion of a substance in the Chlorella medium into the dialysis tubing where a reaction occurred that resulted in the production of a diffusible toxic substance. Of the ingredients present in the Chlorella medium (1), the most likely compound from which a toxic product could arise was D-glucose. The enzyme best known to oxidize D-glucose to an acid and hydrogen peroxide was glucose oxidase. The production of H202 by the action of a similar enzyme in Chondrus was thus indeed possible, An additional clue to the identity of the growth-inhibitory substance was obtained by adding an excess of catalase to a sterile paper disk (i", Difco) which also contained an extract of C. crispus. In the absence of catalase, inhibition was found while the catalase-treated sample showed no inhibition, thus providing the first direct evidence for the involvement of H202 in the growth- inhibitory response. Evidence for Algal Origin of Hexose Oxidase in C. crispus Several genera of marine bacteria have been isolated from the alga Porphyra leucosticta (20) and it Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 is possible that in this alga or other red algae such bacteria may contain enzymes having "glucose oxidase" activity. In order to establish the algal origin for the hexose oxidase, experiments were conducted to eliminate the possibility of microbial contamination of C. crispus as being the enzyme source. Finely ground samples of C. crispus and Euthora cristata as well as sodium phosphate buffered extracts were screened for such contaminating microbes. Growth studies were done using 2216 E medium, a modification of ZoBell’s 2216 medium (21), which consisted of 0.1$ peptone (Difco), 0.1$ yeast extract (Fisher), 1.5$ agar (Difco), and 0.001$ ferric ammonium citrate made to 1 liter with 75$ sea water (Seven Seas Marine Mix, Utility Chemical) and adjusted to a pH between 7.6 and 7.8 with 1.0 N NaOH. Microbial growth resulting after several days both on solid and liquid (agar omitted) media at 5» 18, and 25°C was collected and plated in excess directly on a Chlorella-seeded plate. No growth-inhibitory activity was found associated with the colonies isolated from solid media or pellets from centrifuged liquid culture which indicates the hexose oxidase found in C. crispus is of algal origin. Purification of Chondrus hexose oxidase Chondrus hexose oxidase was purified 117-fold with a recovery of 11$ of the original activity (Table I). Approximately 10 mg of purified enzyme were obtained from Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ro -o 11 85 66 () 100 35 149 650 4,095 8,190 81,420 69,700 49,340 7 29 527 5,520 2 76 Total Total 2,277 Total Specific 84 59 42 542 468 Volume protein carbohy- activity activity Yield Purification hexose of oxidase from Chondrus crispus Sephadex G-200 Stage of Purification (ml) (mg) drate (mg) (Units) (Units/mg protein) 20,000 x g supernatant 690 Ammonium sulfate ppt DEAE-cellulose Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 8 100 g of air-dried fronds. Disc gel electrophoresis of the purified enzyme showed a single band staining with Coomassie blue (Fig. 8). The Chondrus enzyme appeared unaffected by pepsin-trypsin digestion as shown by no loss in biological activity with the Chlorella assay and no alteration in molecular size when examined by gel filtra tion on Sephadex G-200. The digestion step was necessary to remove the red pigment phycoerythrin which persisted as an impurity in preparations of the enzyme. The bulk of this pigment however was removed by DEAE-cellulose chroma tography since it was not adsorbed in the presence of 0.1 M sodium phosphate pH 6.8 and hence washed through the column. Composition and molecular weight of Chondrus hexose oxidase The enzyme showed a carbohydrate content of approximately 70% by the anthrone method using D-galactose as standard and 20% protein by the Lowry method based on bovine serum albumin. Moisture may account for ca. 10% of the weight of the lyophilized enzyme. The carbohydrate composition of Chondrus hexose oxidase was determined on a 1 mg sample of enzyme which was hydrolyzed with 1 ml of 2 N H2S0i|, for k hours in a boiling water bath. Solid BaCO^ was added to the hydrolysate until the pH was approximately 5 and the mixture was then centrifuged. The supernatant and washings from the BaSO^ precipitate were combined and concentrated, and the concentrate was chromatographed along with known sugars on Whatman No. 1 paper in the following Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 Fig. 8. Disc gel electrophoresis of purified Chondrus hexose oxidase. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 0 Reproduced with permission of the copyright owner Further reproduction prohibited without permission. 31 systems: n-butanol:ethanol:water (2:1:1, v/v) (15)» benzene:n-butanol:pyridine:water (l:5*3s3» v/v) (15)» n-butanol:pyridine:water (45:25:40, v/v) (22), and ethyl acetate:pyridine:water (2:1:2, v/v) (22). The chromatograms were sprayed with either aniline, hydrogen phthalate or aniline hydrogen oxalate (22). Galactose and xylose were identified as the principal sugars in the Chondrus enzyme. Galactose appeared to be the predominant sugar, because, based on a galactose standard, the carbo hydrate content of the enzyme was estimated at 70$, whereas, based on a xylose standard, the carbohydrate content cal culated out as 115$* due to a lower color yield from xylose. The amino acid composition was determined with a Spinco Amino Acid Analyzer on a 4.7 mg sample of enzyme which had been hydrolyzed in 6 N HC1 at 110°C for 24 hours (Table II). It appeared rich in aspartic acid, threonine, serine, glutamic acid, glycine, alanine, and valine, and low in the basic amino acids (lysine, histidine, and arginine, the sulfur-containing amino acids (cysteine, methionine) and the aromatic amino acids (tyrosine, phenylalanine). Tryp tophan was not determined. Without corrections for loss or degradation, the total weight of amino acids was calcu lated from the analysis to be 605 ug or ca. 13$ of the sample weight which showed agreement with the low value from the Lowry determination. The glycoprotein nature of Chondrus hexose oxidase was further demonstrated by staining with Alcian Blue Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32 Amino acid composition of Chondrus hexose oxidase •» Amino acid p'lolfe/nig enzyme Molar ratio Lysine 0.0447 5 Histidine 0.0083 1 Ammonia -- - Arginine 0.0247 3 Aspartic acid 0.1689 20 Threonine 0.0851 10 Serine 0.1223 14 Glutamic acid 0.1647 20 Proline 0.0723 9 Glycine 0.1483 18 Alanine 0.1140 14 Half-cystine 0.0264 3 Valine 0.0832 10 Methionine 0.0179 2 Isoleucine 0.0357 4 Leucine 0.0621 8 Tyrosine 0.0198 2 Phenylalanine 0.0459 6 Tryptophan -- - ^Obtained by normalizing values relative to histidine = 1. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33 following cellulose acetate elcetrophoresis (23). By this procedure, both the Chondrus enzyme and glucose oxidase showed a blue band against a pale blue background. Sections from an unstained cellulose acetate strip coinci ding with the stained band were excised and placed in the £-dianisidine-peroxidase mixture (see MATERIALS AND METHODS). The rapid formation of a yellow-orange color indicated the association of "glucose oxidase" activity with the band stained for glycoprotein. Staining a developed strip con taining Chondrus enzyme with Ponceau S (24) resulted in a pink-red band against a pink background having the same mobility as those sections having enzyme activity and staining with Alcian Blue. Chondrus hexose oxidase failed to stain with Schiff's reagent (24) which is not uncommon for glycoproteins rich in carbohydrate (23). An emission spectrum of the enzyme showed copper (with a trace of sodium) to be the only metal present. Using the dithizone method and atomic absorption spectros copy, a value of approximately 0.6% (6 fig/mg enzyme) was obtained. For example, a 9.8 mg sample showed total copper by the dithizone method to be 66 fig and by atomic absorption to be 58 fig. Slight variation was found between determinations for two individually processed samples of purified enzyme (by the dithizone method 0.54-0.67fo Cu). Both methods appeared relative3.y close in agreement. Qualitative determination of flavin adenine dinu Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cleotide (FAD) was done by the method of Pazur and Kleppe (8) which involved treatment of the enzyme at 45°C for 1$ minutes with pyridine. Under such conditions the flavin group of glucose oxidase dissociated. This result was confirmed using glucose oxidase and the split FAD examined by paper chromatography. Using Whatman No. 1 paper with a solvent system consisting of n-butanol:acetone:acetic acid: water (5:2:1:3, v/v) which has been described by Pazur and Kleppe (8), the following Rf values were obtained after exposing the developed chromatograms to ultraviolet light: FAD = 0.10, FMN = 0.24, and treated glucose oxidase = 0.10, (FMN = flavin mononucleotide) A lyophilized 2 mg sample of Chondrus enzyme (pale green in color), treated similarly, showed no trace of FAD. The same result was found with a 5-10 minute treatment in a boiling water bath. The treated Chondrus enzyme in both instances showed no fluorescence unlike denatured glucose oxidase and flavin standards. Supplemental evidence for the absence of FAD was based on a rather featureless visible spectrum which unlike glucose oxidase showed no discernable peaks even at 380 and 460 nm (25) which are characteristic of FAD (Fig. 9). An approximate molecular weight was obtained by gel filtration on Sephadex G-200. A column (2.5 x 43 cm) was equilibrated at 5°C with 0.1 M sodium phosphate pH 6.8 and several proteins of known molecular weight were used as standards (Fig. 10). The elution volume for the Chondrus enzyme corresponded to a molecular weight of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. erdcdwih emiso o h oyih we. ute erdcin rhbtdwihu permission. ithout w prohibited reproduction Further owner. copyright the of ission perm ith w Reproduced Absorbance 0.30 0 4 . 0 0 5 . 0 0.10 0.20 0 0 4 0 0 3 Pig. 9. Visible spectrum of purified Chondrus of purified spectrum Visible 9.Pig. enzyme. Sample was ca. 5 mg/ml in distilled water. distilled in 5mg/ml ca.was Sample enzyme. aeegh (nm) Wavelength 0 0 5 0 0 6 0 0 7 35 36 Fig. 10. Relationship of elution volume to molecular weight for several protein standards and Chondrus hexose oxidase on a column of Sephadex G-200 (2.5 x 4 3 cm), Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6.0 ) ) 0 0 0 0 0 0 , , apoferritin (480,00C 1 5 0 1 5 1 5.5 ) glucose oxidase ( 0 0 0 , Chondrus enzyme (130,000) 6 8 • \gamina• globulin ( ) 0 0 0 , 5.0 albumin ( 2 5 ) Log Molecular Weight ) 0 0 0 , ovalbumin (45,000) 7 0 0 1 7 , 1 3 chymotrypsinogen ( • • myoglobin ( Vs. ribonuclease ( 1.0 2.0 2.5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 c a . 130, 000. Properties of Chondrus hexose oxidase The pH optimum of the enzyme was determined using 0.1 M sodium phosphate buffers ranging in pH from 4.9 to 9.1. The optimum pH appeared to be ca. 6.3 (Fig. 11). The enzyme was found most active at an incu bation temperature of 25°C (Fig. 12). Heat stability of the enzyme was determined by heating for 5 minutes at various temperatures, chilling in an ice bath, and assaying with the o.-dianisidine-peroxidase system (Fig. 13). A sudden drop in activity occurred between 50 and 60°C, Substrate specificity of the enzyme was determined using a number of sugars at a final concentration of 0.1 M (Table III). The substrates most readily oxidized were D-glucose, D-galactose, maltose, cellobiose, and lactose. L-glucose was not oxidized. The five main substrates of the Chondrus enzyme, at a final concentration of 0.1 M, were tested with a partially purified extract of Euthora cristata and also with glucose oxidase (Table IV). The Euthora preparation gave essentially the same results as the Chondrus enzyme but glucose oxidase attacked only D-glucose at a significant rate. In order to determine whether free glucose might be present in the disaccharide samples, 1% solutions of each sugar were chromatographed on Whatman No. 1 paper in either ethyl acetate:pyridine: water (120:50,>40, v/v) or iso-propanol:water (4:1,v/v) and the chromatograms sprayed with aniline hydrogen phthalate Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 Fig. Fig. 11. Effect of pH on enzyme activity (SQ-iun) Jfcj.TATq.ov euutzug Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced Enzyme Activity (Units) 0 2 6 8 i. 2 Efc o nuaintmeaue on temperature incubation of Effect 12. Fig. nye activity. enzyme 0 10 eprtr (°C) Temperature 20 30 40 Reproduced with perm ission of the copyright owner. Further reproduction prohibited w ithout permission. ithout w prohibited reproduction Further owner. copyright the of ission perm with Reproduced Activity (Units) 5 1 2 3 30 Heat odr hexose oxi . e s a id x o e s o x e h s ru hond C f o y t i l i b a t s t a e H . 3 1 . g i F at e ( for 5 n. in m 5 r o f ) C (° re tu ra e p m e T 50 60 erdcdwt pr sin ftecprgtonr Frhrrpouto poiie tot permission. ithout w prohibited reproduction Further owner. copyright the of ission perm with Reproduced Initial Velocity (Units) 2.0 7.0 reaction velocity.reaction Fig. 14. Effect of substrate concentration on concentration ofsubstrate Effect 14.Fig. usrt ocnrto (M) Concentration Substrate 0.10 / °*6' 1/V 0.02 0.2 0.4 100 D-GLUCOSE D-GALACTOSE 200 1/S 0.20 300 2 4 T a b le I I I Substrate specificity of Chondrus hexose oxidase Substrate* Relative Rate D-Glucose 100 D-Galactose 82 Maltose 40 Cellobiose 32 Lactose 22 Glucose 6-phosphate 10 D-Mannose 8 2-Deoxy D-Glucose 8 2-Deoxy D-Galactose 6 D-Fucose 2 D-Glucuronic acid 2 D-Xylose 1 *Sugars not oxidized: L-glucose, D-fructose, D-gluconie acid lactone, Y-galactonolactone, dulcitol, D-gluconic acid, D-arabinose, xylitol; sucrose. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b le IV Comparison of substrate specificity of Euthora and Chondrus enzymes and glucose oxidase Relative Rate E u th o ra Chondrus S u b s t r a t e enzyme enzym e Glucose Oxidase D -G lu c o s e 100 100 100 D-Galactose 95 82 0 M a lto s e 32 40 1 C e llo b io s e 95 32 2 L a c to s e 51 22 0 ^Partially purified sample obtained from DEAE-eellulose column (see METHODS). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 5 followed by heating at 100°C for 5 minutes. A trace of free glucose was detected only in the sample of maltose. The effect of increasing substrate concentra tion on reaction velocity was determined with D-glucose and D-galactose (Fig. 14), and the method of Lineweaver- Burk (26) was used to determine Michaelis constants (Km's). The Km's for D-glucose and D-galactose were 0.00^ M and 0.008 M, respectively. A Km of 0.0025 M for D-glucose was reported for the Iridophycus enzyme (3). The effect of various inhibitors on the Chondrus enzyme was determined (Table V), The most potent inhi bitor was sodium diethyldithiocarbamate, effective at 10**^ M. This compound also inhibited glucose oxidase at this level. The enzyme was also inhibited by sodium cyanide, sodium azide, hydroxylamine hydrochloride, sodium acetate, and sodium pyruvate. The Iridophycus enzyme was reported as being quite sensitive to acetate (3), more so than found with the Chondrus enzyme. Products of Chondrus hexose oxidase The production of hydrogen peroxide in the Chondrus enzyme reaction was shown by omitting peroxidase from the standard assay mixture. When this was done, o-dianisidine was very slowly oxidized to a colored product. Since peroxidase specifically uses ^2°2 'fco oxidize the o-dianisidine, this demonstrates that ^2°2 ^s being produced. Additional evidence was obtained by including an excess of catalase with Chondrus enzyme Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 6 Effect of various inhibitors on Chondrus hexose oxidase •» I n h i b i t o r Concentration (M) Inhibition {%) Sodium diethyl- 1 0 - * 95 dithiocarbamate 10 22 1 o I—1 Sodium cyanide 61 i o " 4 15 Hydroxylamine I Q ' 2 1 00 hydrochloride 1 ( T 3 96 l o ' 4 26 1—1 1 rH O Sodium azide 85 I Q ' 2 78 1 0 ' 3 65 Sodium acetate 1 0 - 1 56 1 0 - 2 13 Sodium pyruvate 10 ~ 4 3 ^Showed no inhibition at 10"^ Ms sodium pyruvate, sodium benzoate, D-gluconic acid, D-gluconic acid lactone and D-glucuronic acid. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. using the Chlorella assay. In the presence of catalase, no inhibition of Chlorella was found. However, in the absence of catalase inhibition occurred. The H202 was decomposed by catalase to water and oxygen both of which are obviously non-toxic to Chlorella. The toxic effect °f h202 to Chlorella was shown by testing this compound at various concentrationss‘a net zone of inhibition of 3.8 cm was found at 10 mg/ml, a 1.6 cm net zone at 1.0 mg/ml, and a net zone of 0.2 cm at 0.1 mg/ml. A paper disk treated with glucose oxidase showed inhibition when tested against Chlorella apparently due to the production of H202 since D-gluconolactone at 10 mg/ml was not inhibitory. The product in addition to H202 formed by the Chondrus hexose oxidase reaction was determined by in cubating the enzyme plus excess catalase in 2 ml of 0,1 I glucose in 0.1 M pH 6.3 sodium citrate buffer at 25°C for ca. 12 hours. Also reacted under the same con ditions was glucose oxidase. Paper chromatography of the reaction mixtures after 12 hours or longer showed the formation of D-gluconolactone from D-glucose and D-galactonolactone from D-galactose (Table VI). The oxidation of D-glucose and D-galactose by Chondrus hexose oxidase can therefore be written as shown in the following reactions: Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 8 T a b le V I Paper chromatography of products from the Chondrus hexose oxidase reaction R-f values t Sample* Solvent A Solvent B Glucose 0.14 0.35 6«*D-Gluconolactone 0.37 0.54 Chondrus enzyme product 0.37 0.46 from glucose Glucose Oxidase product 0.37 0.46 Galactose 0.13 0.42 y-D-Galactonolactone O.32 0.46 Chondrus enzyme product O .32 0.44 from galactose Glucose and galactose were detected with aniline hydrogen phthalate spray (22) and the lactones and oxidation products by spraying with hydroxylamine and ferric chloride (29). tRun on Whatman No. 1 paper. Solvent systems used by Bean et al. (11). A = n-butanol:acetic acids water (52:13:35). B = Phenol:water (80:20)o Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 9 02 D-Glucopyranose 6-D-Gluconolactone CHpOH JHpOH 02 D-Galactopyranose Y-D-Galaetonolaetone Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50 DISCUSSION The substance in Chondrus crispus which is responsible for inhibiting the growth of Chlorella pyrenoidosa has been shown to be hexose oxidase. This enzyme reacts with D-glucose in the Chlorella medium producing the oxidized sugar and hydrogen peroxide. Of the two products formed, H202 has been determined as the actual growth-inhibitory compound. Chondrus hexose oxidase has a wide substrate specificity which includes principally D-glucose, D-galactose, maltose, lactose, and cellobiose. D-glucose and D-galactose are oxidized by this enzyme to the corresponding hexonolactones. Quite similar in properties to the Chondrus enzyme is a hexose oxidase obtained from the red alga Iridophycus flaccidum (3). It likewise produces H2O2 and the aldonic acid (lactone) from D-hexoses (3). Another red alga, Euthora cristata, has been shown to contain a hexose oxidase and as with Chondrus its inhibition to Chlorella is due to In addition to hexose oxidase, various other "glucose oxidases" have been reported. Table VII presents a comparison of these enzymes, their sources, and some of their properties. The sources include red algae, citrus fruits, fungi, bacteria, and honey. The most studied of these enzymes has been glucose oxidase found in the fungi Aspergillus and Penicillium. This enzyme contains 2 FAD per molecular weight of 150,000 to 160,000. Lactose Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. erdcdwt pr sin fte oyih we. ute erdcin rhbtdwihu permission. ithout w prohibited reproduction Further owner. copyright the of ission perm with Reproduced A comparison of properties for various "glucose oxidases" H g •H 3 cd 3 O O ra C 0 a> o 1 - p rH rH W) cd 3 o o O M I I II 1 5 52 dehydrogenase also contains PAD however its molecular weight has not been reported. Regarding substrate specificity, the honey enzyme and glucose oxidase are similar in that both are highly specific for D-glucose. FAD has not been detected in the honey enzyme and no requirement for it has been indicated (4-). A coenzyme other than FAD has not previously been reported for a "glucose oxidase". The finding of a copper-containing enzyme with glucose oxidase activity in Chondrus is new. Whether other "glucose oxidases" contain copper is not known. It is suspected however based on similarity in properties that the Iridophycus and Euthora enzymes also contain copper. The Chondrus enzyme contains approximately 12 gram atoms of copper per mole and apparently no FAD. This large amount of copper can be contrasted to the 1 gram atom per mole reported for galactose oxidase (12), The two enzymes are related in that each contains copper and both produce H202 from oxidation of their substrates. They can be clearly distinguished, however, because substrate oxidation with galactose oxidase occurs at the C-6 position resulting in formation of a dialdehyde from D-galactose while C-l oxidation is found with the Chondrus enzyme with the product being hexonolactone. Like glucose oxidase, galactose oxidase is quite substrate specific, however, the galactose enzyme also oxidizes galactose- containing polysaccharides quite well (13)• Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53 Besides containing a high level of copper, the Chondrus enzyme has been found to contain ca. 70$ carbo hydrate compared to 17$ reported for glucose oxidase (27). The carbohydrate moiety of Chondrus hexose oxidase con sists principally of galactose and xylose while glucose oxidase contains 14$ mannose, 2$ glucosamine, and 1$ galactose (27). The importance of carbohydrate to the Chondrus enzyme's activity has not been determined. Treatment with periodic acid could resolve this point such that loss or reduction in activity would be due to destruction of the carbohydrate portion of the enzyme. The enzymatic activity of glucose oxidase, however, remains unaffected by mild periodate oxidation indicating the carbohydrate residues are probably not involved in the enzyme's active site (27). The glycoprotein nature of Chondrus hexose oxidase has been shown by staining developed strips from cellulose acetate electrophoresis with Alcian Blue (23). A blue band against a pale blue background is shown by the Chondrus enzyme and glucose oxidase. Enzymatic activity in both cases is associated with sections of the strips stained with Alcian Blue. Staining a developed strip containing Chondrus enzyme for protein with Ponceau S (24) gives a pink-red band against a pale pink background which is identical in mobility to those sections staining with Alcian Blue and showing enzyme activity. Chondrus hexose oxidase appears to be a rather Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 stable enzyme such that losses in activity do not occur following pepsin-trypsin digestion, heating at 50°C for 5 minutes, or extensive dialysis against distilled water. Its stability to proteolytic digestion can be explained in part due to its amino acid composition which reflects low levels of tyrosine, phenylalanine, lysine, and arginine. Pepsin would be expected to preferentially hydrolyze at sites adjacent to aromatic amino acids while trypsin would favor cleavage adjacent to lysine or arginine. Perhaps more important than the actual number of these amino acids would be their arrangement or position in the overall structure such that they would or would not be in an accessible location for proteolytic attack. Resistance to pepsin-trypsin digestion is also found with glucose oxidase (28). The Chondrus enzyme's stability to dialysis against distilled water suggests that the copper is tight ly bound to the enzyme. This same type of stability to extensive dialysis is also shown by galactose oxidase (12). The purified Chondrus enzyme is pale green in color which probably results from its high copper content. The5lyophilized enzyme was on occasion difficult to handle because of its very hygroscopic nature. Within a minute after being disconnected from a lyophilizer on a humid day, it changes to a sticky material with a greenish color. This rapid hydration may be related to the large amount of bound copper and possibly the carbohydrate content. A molecular weight of approximately 130,000 is Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. shown by Chondrus hexose oxidase on a column of Sephadex G-200 which had been calibrated with proteins of known molecular weight. This value could differ from the actual molecular weight by 10$ or more. For example, glucose oxidase is slightly retarded on this column and hence shows a molecular weight lower than the actual value. Gel filtration appears to provide at least an estimate of the actual molecular weight. Determination of molecular weight by additional methods will be necessary to confirm this value. A number of compounds have been found to inhibit the Chondrus enzyme, most severely being sodium diethyl- dithiocarbamate effective at 10**^ M. In decreasing order of effectiveness are sodium cyanide, hydroxylamine hydro chloride, sodium azide, sodium acetate, and sodium pyru vate, Diethyldithiocarbamate at lO-^ M has been reported to inhibit completely the enzymatic action of galactose oxidase (12). Also sensitive to this inhibitor to the same extent is glucose oxidase. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56 SUMMARY AND CONCLUSIONS The marine red alga Chondrus crispus has "been found to contain a "glucose oxidase" which is responsible for the observed inhibition to Chlorella pyrenoidosa through its action on glucose in the Chlorella medium which results in the production of the growth-inhibitory compound hydrogen peroxide. An 11$ recovery of activity is realized from a purification procedure involving extraction with 0.1 M sodium phosphate pH 6 .8, n-butanol treatment, DEAE-cellulose chromatography, pepsin-trypsin digestion, and gel filtration on Sephaaex G-200. The enzyme appears unique in having "glucose oxidase" activity while lacking FAD and containing copper instead. Pre liminary studies on Euthora cristata indicate it also contains a hexose oxidase. There are additional experiments which would surely provide a better understanding of the Chondrus enzyme. Such studies as determining the relevance, if any, of the carbohydrate portion to activity, the manner in which the copper is coordinated in the native enzyme, further work on the subunit composition, and molecular weight are but a few examples. Chondrus crispus is found in abundance at Rye Beach, New Hampshire in the inter-tidal zone. Adequate supply of this alga should therefore not present a problem for future study. Euthora cristata however has Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 57 been collected only from the drift and supply has been rather limited. As this alga grows in deeper water the only way by which sufficient material could be collected would be by diving. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 8 BIBLIOGRAPHY 1. Ikawa, M., Ma, D.S., Meeker, G.B., and R.P. Davis, 1969. Use of Chlorella in Mycotoxin and Phycotoxin Research. J. Agr. Food Chem. 17:425-429. 2. Sullivan, J.D. Jr., and M. Ikawa. 1972. Variations in Inhibition of Growth of Five Chlorella Strains by Mycotoxins and Other Toxic Substances. J. Agr. Food Chem. 20:921-922. 3. Bean, R.C. and W.Z. Hassid. 1956. Carbohydrate Oxidase from A Red Alga, Iridophycus flaccidum. J. Biol. Chem. 218:425-436. 4. Schepartz, A.I. and M.H. Subers. 1964. The Glucose Oxidase of Honey. Biochim. Biophys. Acta. 85:228-237. 5. Dowling, J.H. and H.B. Levine. 1956. Hexose Oxidation by An Enzyme System* of Malleomyces pseudomallei. J. Bact. 72:555-560 6. Nishizuka, Y., Kuno, S. and 0. Hayaishi. I960. Lactose Dehydrogenase, A New Flavoprotein, J. Biol. Chem. 235:13-1^. 7. Keilin, D. and E.F. Hartree. 1948. Properties of Glucose Oxidase (Notatin). Biochem. J. 42:221-228. 8. Pazur, J.H. and K. Kleppe. 1964. The Oxidation of Glucose and Related Compounds by Glucose Oxidase from Aspergillus niger. Biochemistry. 3*578—583• Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 59 9. Ruelius, H.W., Kerwin, R.M., and F.W. Janssen. 1968. Carbohydrate Oxidase, A Novel Enzyme from Polyporus ohtusus. Biochim. Biophys. Acta. 167:493-50°. 10. Yoshimura, T. and T. Isemura. 1971. Subunit Structure of Glucose Oxidase from Penicillium amagasakiense. J. Biochem. 69:839-846. 11. Bean, R.C., Porter, G.G., and B.M. Steinberg. 1961. Carbohydrate Metabolism of Citrus Fruits. J. Biol. Chem. 236s1235-1240. 12. Amaral, D., Bernstein, L., Morse, D. and B.L. Horecker. 1963. Galactose Oxidase of Polyporus circinatus: A Copper Enzyme. J. Biol. Chem. 238:2281-2284. 13. Avigad, G., Amaral, D., Asensio, C., and B.L. Horecker, 1962. The Galactose Oxidase of Polyporus circinatus. J* Biol. Chem. 237:2736-2743. 14. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and R.J. Randall. 1951. Protein Measurement with The Folin Phenol Reagent. J. Biol. Chem. 193:263-275. 15. Hodge, J.E. and B.T. Hofreiter. 1962. Determination of Reducing Sugars and Carbohydrates, in Methods in Carbohydrate Chemistry. R. L. Whistler and M.L. Wolfram (Eds.). Vol. I. Academic Press, New York, pp. 24, 389. 16. Ballentine, R. and D.D. Burford. 1957. Determination of Metals, in Methods in Enzymology. S.P. Colowick and N.O. Kaplan (Eds,). Vol. III. Academic Press, New York. pp. 1024, IOO3. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 17. Williams, D.E. and R.A. Reisfeld. 1964. Arm. N.Y. Acad. Sci. 121:373. 18. Huggett, A.S.G. and D.A. Nixon. 1957. Use of Glucose Oxidase, Peroxidase, and o-Dianisidine in Determination of Blood and Urinary Glucose. Lancet. 2:368-370. 19. Leibo, S.P. and R.F. Jones. 1964. Freezing of The Chromoprotein Phycoerythrin from The Red Alga Porphyridium cruentum. Arch. Biochem. Biophys. 106:78-88. 20. Tsukidate, J. 1971. Microbiological Studies on Porphyra Plant. Bull. Soc. Scientific Fisheries. 37:376-379. 21. ZoBell, C.E. 1944. Studies on'ivlarine Bacteria. J. Mar. Res. 4:42-75. 22. Block, R.J., Durrum, E.L., and G. Zweig. 1955. Paper Chromatography and Paper Electrophoresis. Academic Press, New York. pp. 128, 133. 23. Wardi, A.H. and W.S. Allen. 1972. Alcian Blue- Staining of Glycoproteins. Anal. Biochem. 48:621-623. 24. Smith, I. i960. Chromatographic and Electrophoretic Techniques. Interscience Publishers, New York, pp. 16, 69. 25. Bodmann, 0. and M. Walter. 1965. Die Glucose-Oxydasen Aus Penicillium notatum (Notatin) und Aspergillus niger (Nigerin). Biochim. Biophys. Acta. 110:496-506. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 61 26. Lineweaver, H. and D. Burk. 193^. The Determination of Enzyme Dissociation Constants. J. Am. Chem. Soc. 56:658-666. 27. Pazur, J.H., Kleppe, K., and E.M. Ball. 1963. The Glycoprotein Nature of Some Fungal Carbohydrases. Arch. Biochem. Biophys. 103:515-518. 28. Bentley, R. 1955. Glucose Aerodehydrogenase (Glucose Oxidase), in Methods in Enzymology. S.P. Colowick and N.O. Kaplan (Eds.). Vol. I. Academic Press, New York. pp. 3^°-3^5. 29. Abdel-Akher, M. and F. Smith. 1951. The Detection of Carbohydrate Esters and Lactones after Separation by Paper Chromatography. J. Am. Chem. Soc. 73:5859-5860. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 62 APPENDIX During the course of this work a number of com pounds have been tested against strains of Chlorella. The results of these tests are presented in the following tables. Table IA which has been taken directly from a paper by Sullivan and Ikawa (2) shows the activity of some toxins and inhibitors on Chlorella strains. It is apparent from these results that strains of Chlorella differ in sensitivity to certain compounds. Perhaps the most sensitive strains are UNH and 395 of C. pyrenoidosa. For a more lengthy discussion of this material the reader is referred to the original paper (2). The effect of several pesticides on Chlorella strains is shown in Table IIA. As found in Table IA, there exists here also a variation in sensitivity among Chlorella strains in this case to pesticides. C. pyrenoi dosa (UNH strain) and C. vulgaris are not inhibited by these pesticides when tested at 1 mg/ml. In Table IIIA are found the results of testing a variety of miscellaneous compounds and antibiotics against C. pyrenoidosa (UNH strain). A number of steroidal compounds have been also tested although none have proven inhibitory to Chlorella (Table IVA). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 0 0 4 0 14 Tr 20' Tr 28 8° 0 0 21 8C 0 0 0 0 0 0 0 21 0 06° 0 0 6 6 6 0 0 0 0 13 3 9 C 2 Tr 25 Tr 3§ 18 C. C. pyrenoidosa Diameter of Net Zone of Inhibition, 0 0 0 /+ 0 0 Tr1 17 10 i9f Tr 25 40° UNH 395 251 252 vulgaris C. Table IA 1 1 1 0.01 1 0 1 0.01 0.1 0.1 1 1 0.1 0.01 0.1 Cone. Water Ethanol Water Water Ethanol Ethanol DMSO 1 Ethanol DMSO 1Ethanol 0 0 0 0 0 DMSO DMSOd DMSO Solvent (mg/ml) Ethanol Ethanol Ethanol scirpenol Gramicidin J Aflatoxin G2 Emodin Aflatoxin G-^ Digitonin Diacetoxy- Aflatoxin B^A6 Aflatoxin B3 DMSO 1 Aflatoxin Acrylic acid Compound Growth-Inhibitory Activity of Some Toxins and Inhibitors on Chlorella Strainsa Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 C. vulgaris C. of Inhibition, mm13 Inhibition, of 1 for gramicidin + 36 252 ). ). eA synthetic 1 9 6 9 14° 13° 251 pyrenoidosa 0 0 0 0 395 Tr C, Diameter of Net Zone Net of Diameter 0 0 0 0 0 0 15° UNH 1 1 1 0.1 0 0 0 0 Cone. (fflg/ml) Table IATable (continued) Ethanol Ethanol Solvent Ethanol Ethanol for zearalenone, when all compounds were tested at 1 mg7ml. Diameter 15+1 (F-2) Sterile disks (Difco Laboratories) of 0.6-cm diameter were used on buffered agar Zearalenone® Compound Rubratoxin B Rubratoxin Kainic acid Kainic J, J, and of disk subtracted from total diameter oftoxyscirpenol, inhibition zone. small zones Chlorellaat cWeak complete growth growth This inhibitionof extendingChlorella zone had were beenanalog3*5also mm ignoredbeyondobservedof aflatoxin in tration,the within previousdisk was Washington,thework zone observed crystals (Ikawa D.C. with wereet al., DMSO kindly was as kindly fTrace solvent.supplied suppliedindicates by C.J. bya Mirocha,netJ.V. zone Rodricks, Universityof 3 mm Food or andofless. Minnesota, Drug Adminis St. ®F-2 Paul, Minn. pyrenoidosa (UNH strain), on which the most assays were denserrun, weregrowth. This often made reading the zones difficult. In the case of diace- where partial growth had occurred. DMSO = dimethylsulfoxide. A zone of weak plates. minations Values ofeach inhibitionbility run inof duplicate response,represent with thean averagemeantwo disks and ofstandard perat leastplate. deviationthree separate from To illustrate thedeter mean inthe thevaria case of C. was observed within the inhibition zone which was surrounded by a background of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 0 0 0 0 0 0 0 0 C. vulgarisC. Inhibition, Inhibition, mm 3 3 2 0 6 2 6 3 2 Ur Ur Carolina 15-2070 C. C. pyrenoidosa Diameter of Net Zone of 0 0 0 0 0 0 0 0 0 UNH Table IIA 1 1 1 1 0.1 1 0.1 1 1 1 1 Cone. Cone. * (mg/ml) Effect Effect Several of Pesticides on Chlorella Strains ^Solvent = 95$ ethanol Chlordane Sevin Aldrin Methoxychlor Lindane Compound Toxaphene DDT Dieldrin Endrin Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 0 7 5 0 0 Zone Zone of Inhibition, mm pyrenoidosa (UNH strain) 1 1 1 6 1 1 1 0 1 0 1 Cone. (mg/ml) Diameter of Net Solvent Ethanol DMSO Ethanol 1Ethanol 1 2 0 Water 1 Ethanol 1 0 Ethanol 1 0 Ethanol Ethanol Ethanol Ethanol Effect of Various Compounds on Chlorella Compound Albamycin acid NaChloramphenicol Water Water 1 1 0 0 Rotenone Achromycin HC1 • BacitracinCoumarin Dicumarol Water 1 0 Streptomycin sulfate Water Oligomycin Amobarbital Antimycin A Penicillin G, K salt Water. if-Hydroxy coumarin if-Hydroxy Valinomycin i Monsensin jD-Benz oquinone jD-Benz Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 67 Tab3.e IVA Effect of Steroidal Compounds on Chlorella pyrenoidosa * Compound Diameter of Net Inhibition Zone, mm 8,24, 5“-cholestadien- 0 4,4,14a-trimethyl 3 S-cl ^a-cholesta-3-one 0 5«-chclestan=3“One 0 Stigmasterol 0 Progesterone 0 Testosterone 0 Pregnenolone 0 Deoxycorticosterone 0 Sitosterol 0 Ergosterol 0 Betulin 0 Hydrocortisone acetate 0 Androsterone 0 Cortisone acetate 0 Cholesterol 0 17^-estradiol 0 Estriol 0 Ouabain 0 (UNH strain) Compounds tested at 1 mg/ml in ethanol. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.